Abstract
Field emission scanning electron microscopy (FESEM) can provide high-resolution three-dimensional surface imaging of many biological structures, including nuclear envelopes and nuclear pore complexes (NPCs). For this purpose, it is important to preserve NPCs as close as possible to their native morphology, embedded in undamaged nuclear membranes. We present optimized methodologies for FESEM imaging in a cell-free reconstitution system and for the direct visualization of mammalian cell nuclei. The use of anchored chromatin templates in the cell-free system is particularly advantageous for imaging fragile intermediates inhibited at early stages of assembly. Our new method for exposing the surface of mammalian nuclei results in an unprecedented quality of NPC images, avoiding detergent-induced and physical damage. These new methodologies pave the way for the combined use of FESEM imaging with biochemical and genetic manipulation, in cell-free systems and in mammalian cells.
| Original language | English |
|---|---|
| Pages (from-to) | 407-413 |
| Number of pages | 7 |
| Journal | Structure |
| Volume | 20 |
| Issue number | 3 |
| DOIs | |
| State | Published - 7 Mar 2012 |
| Externally published | Yes |
Bibliographical note
Funding Information:The authors thank Eugenia Klein for advice on the generation of stereo pair images and Rita Gruber for help with the assembly diagram. We also thank Ulrike Kutay for the gift of anti-Nup107 antibodies, Lauren Weil and Beatriz Fontoura for the gift of mouse lung fibroblasts, Dana Savulescu for COS-1 cells, and Lena Kliouchnikov for HeLa cells. This work was supported by grants from the Israel Science Foundation (1072/10) and the Russell Berrie Nanotechnology Institute - Technion to A.H.